The long-term objectives of this project are to determine the pathological mechanism of osteonecrosis of the jaw (ONJ) and to develop preventive and therapeutic modalities. ONJ is an oral complication experienced by some patients treated with nitrogen-containing bisphosphonates (BPs) and more recently with humanized anti- RANKL antibody. Confirmed clinical ONJ cases are characterized by the prolonged exposure of partially necrotic jawbone facilitated by abnormal wound healing of oral mucosa. Unlike the well-established pharmacological effect on osteoclasts, the role of BP on oral mucosa has not been fully elucidated. A number of studies have demonstrated that BP affects the healing of intestinal ulceration, the development of peritonitis, and the activation of peripheral blood innate and acquired immunity. The administration routes (PO, IP and IV, respectively) allow the direct exposure of BP to these tissues, prior to rapid adsorption of BP to bone or excretion. However, the source of free BP affecting the oral mucosa has not been identified. To elucidate the mechanism of oral mucosa abnormality associated with ONJ, we have hypothesized that BP pre-adsorbed on the jawbone can be removed by osteoclasts and transiently released to the oral mucosa tissue. We envision that the amount of released BP may depend on the degree of osteoclastogenesis localized at the surface of alveolar bone interfacing oral mucosa; and upon reaching a critical concentration, the released BP may directly affect oral mucosa resident cells and adversely influence the healing of oral mucosa wound. To address this hypothesis, this exploratory R21 project proposes the synthesis and application of novel BP-based probes capable of delivering a Frster resonance energy transfer (FRET)-quenched reporter function to osteoclasts, where the probes will generate a fluorescent signal only in response to osteoclast-derived cathepsin K (CatK) activity which causes separation of emitter and quencher. Novel molecular probes consisting of a short CatK- cleavable peptide bridging a fluorescent dye and a quencher will be attached to a biologically active or inactive BP scaffold through unique BP-linker chemistry developed at USC. The silent BP-CatK-FRET probe will be strongly adsorbed to bone or synthetic calcium phosphate, but osteoclastic CatK will remove the quencher and activate the fluorescent signal. BP-CatK-FRET activation by osteoclasts will be characterized in vitro using synthetic apatite-coated plates and mouse osteoclasts (correlating fluorescent signal with resorption pit area, Rap1a prenylation and osteoclast apoptosis). The proposed probes will also be powerful tools for in vivo evaluation to establish the critical link between BP mobilized by osteoclasts and oral mucosa resident cells. They will also be useful to assess the role of legacy BP drugs in determining ONJ risk and to examine future replacement therapy approaches. In implementing this project, the research teams of McKenna (USC) and Nishimura (UCLA) will leverage their complementary strengths in BP probe design and chemistry (USC) and expertise in bone biology models (UCLA).